Solid dose delivery vehicle and methods of making same

ABSTRACT

The present invention encompasses a solid dose delivery vehicle for ballistic administration of a bioactive material to subcutaneous and intradermal tissue, the delivery vehicle being sized and shaped for penetrating the epidermis. The delivery vehicle further comprises a stabilizing polyol glass loaded with the bioactive material and capable of releasing the bioactive material in situ. The present invention further includes methods of making and using the solid dose delivery vehicle of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 10/652,212,filed Aug. 29, 2003; which is a continuation of application Ser. No.10/376,136, filed Feb. 27, 2003, now U.S. Pat. No. 6,893,657; which is acontinuation of application Ser. No. 09/945,180, filed Aug. 31, 2001,now U.S. Pat. No. 6,565,871; which is a continuation of application Ser.No. 09/628,380, filed Aug. 1, 2000, now U.S. Pat. No. 6,331,310; whichis a continuation of application Ser. No. 08/349,029, filed Dec. 2,1994, now U.S. Pat. No. 6,290,991. The disclosure of each of these isincorporated by reference herein as if put forth below in full.

FIELD OF THE INVENTION

The present invention relates generally to solid dose vehicles fordelivery of bioactive materials and, more specifically, to solid dosedelivery vehicles comprising a stabilizing polyol and a bioactivematerial. Methods of their making and uses thereof are also provided.

BRIEF SUMMARY OF THE INVENTION

Solid dose delivery of bioactive materials to mucosal, dermal, ocular,subcutaneous, intradermal and pulmonary tissues offers severaladvantages over previous methods such as topical applications ofliquids, transdermal administration via so-called “patches” andhypodermic injection. In the case of injection, solid dose delivery canreduce the risk of infection by eliminating the use of needles andsyringes, provide for more accurate dosing than multidose vials, andminimize or eliminate the discomfort which often attends hypodermicinjection. Several solid dose delivery systems have been developedincluding those utilizing transdermal and ballistic delivery devices.

Topical delivery is utilized for a variety of bioactive materials suchas antibiotics for wound healing. These topical ointments, gels, creams,etc. must be frequently reapplied in order to remain effective. This isparticularly difficult in the case of burn wounds.

Devices used for administering drugs transdermally usually compriselaminated composites with a reservoir layer of drug with the compositebeing adhered to the skin, i.e., transdermal patch, such as described inU.S. Pat. No. 4,906,463. However, many drugs are not suitable fortransdermal delivery, nor have transdermal drug release rates for thosecapable of transdermal delivery been perfected.

Subdermal implants have also been formulated for slow release of certainpharmaceutical agents for extended periods of time such as months oryears. A well-known example is the Norplant® for delivery of steroidhormones. Such implants are usually constructed of an inner, drug-filledcore which is relatively permeable to the drug and an outer matrix whichis relatively impermeable to the drug. Both inner core and outer matrixare generally formed from polymers. The implants release drugs bydissolution of the drug in the inner core and slow release across theouter matrix. The inner core may substantially dissolve over time,however, in devices currently in use, the outer matrix does notdissolve. Implants are placed subcutaneously by making an incision inthe skin and forcing the implants between the skin and the muscle. Atthe end on their use, if not dissolved, these implants are surgicallyremoved. U.S. Pat. No. 4,244,949 describes an implant which has an outermatrix of an inert plastic such as polytetrafluoroethylene resin. PCT/GB90/00497 describes slow release vitreous systems for formation ofimplantable devices. These implants are bioabsorbable and need not besurgically removed. However, insertion is by surgical means. Moreover,these devices may be limited in the type of bioactive material that canbe incorporated. In the case of polymeric implants, bioactive materialsthat cannot withstand organic solvents are not suitable for use. In thecase of vitreous systems, bioactive materials that cannot withstand theelevated temperatures necessary to form the implants are unsuitable foruse. In all cases, bioactive materials that are unstable at bodytemperature, particularly over long time periods, are unsuitable foruse.

A variety of formulations have been provided for administration inaerosolized form to mucosal surfaces, particularly “by-inhalation”(naso-pharyngeal and pulmonary). Compositions for by-inhalationpharmaceutical administration generally comprise a liquid formulation ofthe pharmaceutical agent and a device for delivering the liquid inaerosolized form. U.S. Pat. No. 5,011,678 describes suitablecompositions containing a pharmaceutically active substance, abiocompatible amphophilic steroid and a biocompatible (hydro/fluoro)carbon propellant. U.S. Pat. No. 5,006,343 describes suitablecompositions containing liposomes, pharmaceutically active substancesand an amount of alveolar surfactant protein effective to enhancetransport of the liposomes across a pulmonary surface.

One drawback to the use of aerosolized formulations is that maintenanceof pharmaceutical agents in aqueous suspensions or solutions can lead toaggregation and loss of activity and bioavailability. The loss ofactivity can be partially prevented by refrigeration; however, thislimits the utility of these formulations. This is particularly true inthe case of peptides and hormones. For instance, synthetic gonadotropinreleasing hormone (GnRH) analogs, such as the agonist nafarelin or theantagonist ganirelex are designed for high potency, increasedhydrophobicity and membrane binding. The compounds have sufficienthydrophobic character to aggregate in aqueous solution and to form anordered structure that increases in viscosity with time. Thusbioavailability in nasal or pulmonary formulations may be prohibitivelylow. The use of powdered formulations overcomes many of these drawbacks.The requisite particle size of such powders is 0.5-5 microns in order toattain deep alveolar deposition in pulmonary delivery. Unfortunately,powders of such particle size tend to absorb water and clump and thusdiminish deposition of the powder in the deep alveolar spaces. Althoughpowders with larger particle size are suitable for delivery to thenaso-pharynx region, the tendency of powders to clump decreases theavailable particle surface area for contact with, and absorptionthrough, these membranes. Devices which disaggregate clumps formed byelectrostatic interactions are currently in use (e.g., the Turbohaler);however, these do not disaggregate moisture induced clumps and it wouldbe advantageous to have powders which do not absorb moisture and clumpand thus increase the effective pulmonary concentration of the drug.

Solid dose delivery vehicles for ballistic, transdermal, administrationhave also been developed. For example, in U.S. Pat. No. 3,948,263, aballistic animal implant comprised of an exterior polymeric shellencasing a bioactive material is described for veterinary uses.Similarly, in U.S. Pat. No. 4,326,524, a solid dose ballistic projectilecomprising bioactive material and inert binder without an exteriorcasing is disclosed. Delivery is by compressed gas or explosion. Gelatincovered tranquilizing substances carried by ballistic projectiles forimplant are also described in U.S. Pat. No. 979,993.

The above-described ballistic devices, however, are suited to largeanimal veterinary applications due to their relatively large size, onthe order of millimeters. Ballistic delivery at the cellular level hasalso been successful. The general principle of ballistic administrationis the use of a supersonic wavefront, created by the release ofcompressed gas, to propel the particles contained in an adjoiningchamber. For example, nucleic acids adsorbed on tungsten microprojectileparticles have been successfully delivered to living epidermal plantcells. See Klein, Nature 327:70-73 (1987). A better controlled device isthe particle inflow gun (PIG). Vain et al. (1993) Plant Cell, Tissue andOrgan Culture 33:237-246. Devices have been described which fire ampulescontaining medication using gas pressure. U.S. Pat. No. 4,790,824; andPCT/GB 94/00753. Several devices that inject fluids have also beendescribed. U.S. Pat. Nos. 5,312,335 and 4,680,027. There are fewexisting formulations suitable for ballistic delivery. Powderformulations of pharmaceuticals in their present form are unsuitable forballistic administration. Particles of available powder forms aregenerally irregular, varying in size, shape and density. This lack ofuniformity leads to powder deposit and loss at the skin surface duringadministration, as well as problems in control and consistency of thedepth of delivery to subcutaneous and intradermal tissues.

Thus it would be advantageous to provide solid dose drug deliveryvehicles of defined size, shape and density, to ensure more uniformdistribution. Additional benefits would accrue if the shape of thevehicle could be controlled to facilitate or control penetration of theepidermis and hard layers of the skin. Small delivery vehicle size,preferably coupled with high momentum delivery, would also increase thecomfort of administration and minimize tissue damage. The manufacture ofsuch a solid dose delivery vehicle should be such that neither thedelivery vehicle nor the bioactive substance being delivered is damagednor its efficacy decreased. Furthermore, the bioactive substance shouldremain stable when loaded within or on the vehicle so that efficaciousadministration can be achieved, and to facilitate storage of the loadeddelivery vehicle. Manufacture of the solid dose delivery vehicle and itsloading with bioactive material and the administration of the vehicleshould also be relatively simple and economical.

All references cited herein are hereby incorporated by reference.

SUMMARY OF THE INVENTION

The present invention encompasses a solid dose delivery vehicle suitablefor therapeutic administration of a wide variety of substances,comprising a stabilizing polyol and a bioactive material. Preferredbuffers, adjuvants and additional stabilizers are also provided. Thedelivery vehicle can be sized and shaped for a variety of modes ofadministration.

The invention further includes a solid dose delivery vehicle comprisingan outer portion comprising a water soluble glassy and/or polymericmaterial having a hollow compartment therein, and an inner portionresiding in the compartment, the inner portion comprising at least onestabilizing polyol and a therapeutically effective amount of at leastone bioactive substance.

The invention also encompasses methods of delivering a bioactivematerial by providing a solid dose delivery vehicle described above andadministering the vehicle to the tissue. Administration can be bymucosal, oral, topical, subcutaneous, intradermal and by-inhalation.

The invention further encompasses methods of making the solid dosedelivery vehicle. The stabilizing polyol, bioactive material and anyother components are mixed and processed by a wide variety of methods,including milling, spray drying, freeze drying, air drying, vacuumdrying, fluidized-bed drying, coprecipitation and critical fluidextraction. The dried components can be heated to fluidize the glasswhich can then be drawn or spun into solid or hollow fibers. The driedcomponents can also be mixed in aqueous or organic solutions and dried,such as by spray drying, freeze drying, air-drying, vacuum drying,fluidized-bed drying, co-precipitation and critical fluid extraction.

The invention further provides methods of making vehicles suitable forslow or pulsatile release of bioactive substances. The methods includecombining bioactive material in solid solutions in stabilizingglass-forming polyol and other glass formers with dissolution ordegradation rates slower than that of the glass-forming polyol, andprocessing the components as described above. The ratio of materials canbe controlled so as to provide a wide range of narrowly defined releaserates. The coformulations of stabilizing polyol and other water-solubleand/or biodegradable glasses, plastics and glass modifiers producedthereby are also encompassed by the present invention.

The invention further provides methods of making delivery vehicles ofglasses of hydrated carbohydrates hydrates with increased T_(g) and thecompositions obtained thereby. The method comprises adding a modifier,preferably a protein, in an amount sufficient to elevate the T_(g), tothe carbohydrate and processing according to a method described herein.The modifier may be an inert material or may be the bioactive material.The product obtained may be combined with stabilizing polyols with aT_(g) less than that of the modified carbohydrate to form a slow and/orpulsatile delivery system.

The vehicles and methods of the invention also encompass vehicles whichcomprise fibers, spheres, particles and needles. Preferably thesevehicles are fibers, spheres, particles and needles. The vehicles can beeither microscopic or macroscopic.

A wide variety of bioactive materials are suitable for use in accordwith the present invention, including, but not limited to, therapeuticand prophylactic agents. The delivery vehicle and methods of the presentinvention provide for a variety of dosing schemes for delivery of thebioactive material and are suitable for both veterinary and humanapplications.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting typical particle size distribution ofmicronized trehalose glass powder suitable for administration byinhalation.

FIG. 2A is a graph depicting the sharp particle size distribution fortrehalose/MWPB glass powder. FIG. 2B is a graph depicting the wetting ofvarious trehalose/MWPB glass powders after storage at ambienttemperature and relative humidities.

FIG. 3 is a graph depicting the sharp particle size distribution fortrehalose glass powder obtained by spray-drying in a Lab-plant spraydryer.

FIG. 4 is a graph depicting a comparison of the sharp particle sizedistribution for trehalose glass powders prepared with two differentspray-dryers (Lab-plant and Buchi, as indicated).

FIG. 5 is a graph depicting the release of a dye (Mordant Blue 9) fromcoformulated melt glasses of trehalose octaacetate (TOAC) and raffinoseundecaacetate (RUDA).

FIG. 6A is a graph depicting the resistance of horseradish peroxidase toacetone effected by drying the enzyme with trehalose. FIG. 6B is a graphdepicting the resistance of alkaline phosphatase to acetone effected bydrying the enzyme with trehalose.

FIG. 7 is a graph depicting the effect of a glass modifier on the T_(g)of Trehalose.

FIG. 8 is a graph depicting the effect of a glass modifier on the T_(g)of maltose.

DETAILED DESCRIPTION OF THE INVENTION

The present invention comprises a solid dose delivery vehicle formucosal, oral, topical, subcutaneous and intradermal and by-inhalationadministration comprising a stabilizing polyol and a therapeuticallyeffective amount of a bioactive material. By “solid dose” as usedherein, is meant that a bioactive material delivered by the vehicle isin solid rather than liquid or aqueous form. It has now been found thatstabilizing polyols can be formulated into solid vehicles suitable fordrug delivery. These stabilizing polyols have been found to beparticularly useful where otherwise denaturing conditions would renderimpossible the formulation of solid dosage forms of bioactive materials.In particular, such conditions include elevated temperatures and thepresence of organic solvents.

The compositions exist as solid solutions of the bioactive material instabilizing polyol-glass continuous phases. Previous studies have shownthat in this form the product is resistant to high temperatures with theexact temperatures depending on the stabilizing polyol used. Thus, thecompositions can be processed as glassy melts for brief periods withoutbeing damaged by the processing. In the same way, the stabilizing polyolcontaining the product would be resistant to damage during sinteringwith nitrate and/or carboxylate and/or derivatized carbohydrate and/orother glass-forming substances.

Examples of types of bioactive materials that may be used in the vehicleand methods of the invention include any pharmaceutical agents,including, but not limited to, antiinflammatory drugs, analgesics,antiarthritic drugs, antispasmodics, antidepressants, antipsychotics,tranquilizers, antianxiety drugs, narcotic antagonists, antiparkinsonismagents, cholinergic agonists, chemotherapeutic drugs, immunosuppressiveagents, antiviral agents, antibiotic agents, appetite suppressants,antiemetics, anticholinergics, antihistaminics, antimigraine agents,coronary, cerebral or peripheral vasodilators, hormonal agents,contraceptives, antithrombotic agents, diuretics, antihypertensiveagents, cardiovascular drugs, opioids, and the like.

Suitable bioactive materials also include therapeutic and prophylacticagents. These include, but are not limited to, any therapeuticallyeffective biological modifier. Such modifiers include, but are notlimited to, subcellular compositions, cells, bacteria, viruses andmolecules including, but not limited to, lipids, organics, proteins andpeptides (synthetic and natural), peptide mimetics, hormones (peptide,steroid and corticosteroid), D and L amino acid polymers,oligosaccharides, polysaccharides, nucleotides, oligonucleotides andnucleic acids, including DNA and RNA, protein nucleic acid hybrids,small molecules and physiologically active analogs thereof. Further, themodifiers may be derived from natural sources or made by recombinant orsynthetic means and include analogs, agonists and homologs. As usedherein “protein” refers also to peptides and polypeptides. Such proteinsinclude, but are not limited to, enzymes, biopharmaceuticals, growthhormones, growth factors, insulin, monoclonal antibodies, interferons,interleukins and cytokines. Organics include, but are not limited to,pharmaceutically active chemicals with amino, amino and guanidinogroups. Suitable steroid hormones include, but are not limited to,estrogen, progesterone, testosterone and physiologically-active analogsthereof. Numerous steroid hormone analogs are known in the art andinclude, but are not limited to, estradiol, SH-135 and tamoxifen. Manysteroid hormones such as progesterone, testosterone and analogs thereofare particularly suitable for use in the present invention as they arenot absorbed transdermally and, with the exception of a few analogs, aredestroyed upon oral administration by the so-called hepatic first passmechanism. Therapeutic agents prepared by the methods described hereinare also encompassed by the invention. As used herein, “nucleic acids”includes any therapeutically effective nucleic acids known in the artincluding, but not limited to DNA, RNA and physiologically activeanalogs thereof. The nucleotides may encode single genes or may be anyvector known in the art of recombinant DNA including, but not limitedto, plasmids, retroviruses and adeno-associated viruses. Preferably, thenucleotides are administered in the powder form of the solid dosevehicle.

Compositions containing prophylactic bioactive materials and carrierstherefore are further encompassed by the invention. Preferablecompositions include immunogens such as vaccines. Suitable vaccinesinclude, but are not limited to, live and attenuated viruses, nucleotidevectors encoding antigens, bacteria, antigens, antigens plus adjuvants,haptens coupled to carriers. Particularly preferred are vaccineseffective against diphtheria, tetanus, pertussis, botulinum, cholera,Dengue, Hepatitis A, C and E, hemophilus influenza b, herpes virus,Hylobacterium pylon, influenza, Japanese encephalitis, meningococci A, Band C, measles, mumps, papilloma virus, pneumococci, polio, rubella,rotavirus, respiratory syncytial virus, Shigella, tuberculosis, yellowfever and combinations thereof. Vaccines may also be produced bymolecular biology techniques to produce recombinant peptides or fusionproteins containing one or more portions of a protein derived from apathogen. For instance, fusion proteins containing the antigen ofinterest and the B subunit of cholera toxin have been shown to induce animmune response to the antigen of interest. Sanchez et al. (1989) Proc.Natl. Acad. Sci. USA 86:481-485.

Preferably, the immunogenic composition contains an amount of anadjuvant sufficient to enhance the immune response to the immunogen.Suitable adjuvants include, but are not limited to, aluminum salts,squalene mixtures (SAF-1), muramyl peptide, saponin derivatives,mycobacterium cell wall preparations, monophosphoryl lipid A, mycolicacid derivatives, nonionic block copolymer surfactants, Quil A, choleratoxin B subunit, polyphosphazene and derivatives, and immunostimulatingcomplexes (ISCOMs) such as those described by Takahashi et al. (1990)Nature 344:873-875. For veterinary use and for production of antibodiesin animals, mitogenic components of Freund's adjuvant can be used. Aswith all immunogenic compositions, the immunologically effective amountsof the immunogens must be determined empirically. Factors to beconsidered include the immunogenicity, whether or not the immunogen willbe complexed with or covalently attached to an adjuvant or carrierprotein or other carrier, route of administration and the number ofimmunizing doses to be administered. Such factors are known in thevaccine art and it is well within the skill of immunologists to makesuch determinations without undue experimentation.

The present invention encompasses compositions and methods of making thecompositions. Although singular forms may be used, more than one polyol,more than one biological substance and more than one inhibitor of theMaillard reaction may be present. Determination of the effective amountsof these compounds is within the skill of one in the art.

As used herein, the term “carbohydrates” includes, but is not limitedto, monosaccharides, disaccharides, trisaccharides, oligosaccharides andtheir corresponding sugar alcohols, polyhydroxy compounds such ascarbohydrate derivatives and chemically modified carbohydrates,hydroxymethyl starch and sugar copolymers (Ficoll). Both natural andsynthetic carbohydrates are suitable for use herein. Syntheticcarbohydrates include, but are not limited to, those which have theglycosidic bond replaced by a thiol or carbon bond. Both D and L formsof the carbohydrates may be used. The carbohydrate may be non-reducingor reducing. Suitable stabilizing polyols are those in which a bioactivematerial can be dried and stored without losses in activity bydenaturation, aggregation or other mechanisms. Prevention of losses ofactivity can be enhanced by the addition of various additives such asinhibitors of the Maillard reaction as described below. Addition of suchinhibitors is particularly preferred in conjunction with reducingcarbohydrates.

Reducing carbohydrates suitable for use in the present invention arethose known in the art and include, but are not limited to, glucose,maltose, lactose, fructose, galactose, mannose, maltulose, iso-maltuloseand lactulose.

Non-reducing carbohydrates include, but are not limited to, non-reducingglycosides of polyhydroxy compounds selected from sugar alcohols andother straight chain polyalcohols. Other useful carbohydrates includeraffinose, stachyose, melezitose, dextran, sucrose and sugar alcohols.The sugar alcohol glycosides are preferably monoglycosides, inparticular the compounds obtained by reduction of disaccharides such aslactose, maltose, lactulose and maltulose. The glycosidic group ispreferably a glucoside or a galactoside and the sugar alcohol ispreferably sorbitol (glucitol). Particularly preferred carbohydrates aremaltitol (4-O-β-D-glucopyranosyl-D-glucitol), lactitol(4-O-β-D-galactopyranosyl-D-glucitol), iso-maltulose, palatinit (amixture of GPS, α-D-glucopyranosyl-1-6-sorbitol and GPM), andα-D-glucopyranosyl-1-6-mannitol, and its individual sugar alcohols,components GPS and GPM.

Preferably, the stabilizing polyol is a carbohydrate that exists as ahydrate, including trehalose, lactitol and palatinit. Most preferably,the stabilizing polyol is trehalose. It has now been found that,surprisingly, solid dose compositions containing sugar hydrates liketrehalose lack the “stickiness” or “tackiness” of solid dose formscontaining other carbohydrates. Thus, for manufacture, packaging andadministration, trehalose is the preferred carbohydrate. Trehalose,α-D-glucopyranosyl-a-D-glucopyranoside, is a naturally occurring,non-reducing disaccharide which was initially found to be associatedwith the prevention of desiccation damage in certain plants and animals'which can dry out without damage and can revive when rehydrated.Trehalose has been shown to be useful in preventing denaturation ofproteins, viruses and foodstuffs during desiccation. See U.S. Pat. Nos.4,891,319; 5,149,653; 5,026,566; Blakeley et al. (1990) Lancet336:854-855; Roser (July 1991) Trends in Food Sci. and Tech. 166-169;Colaco et al. (1992) Biotechnol. Internat., 345-350; Roser (1991)BioPharm. 4:47-53; Colaco et al. (1992) Bio/Tech. 10:1007-1011; andRoser et al. (May 1993) New Scientist, pp. 25-28.

It has also now been found, surprisingly, that the glass transitiontemperature (T_(g)) of trehalose can be elevated by the addition ofglass modifiers. Preferably the glass modifiers are proteins thatcomprise 0.002-50% of the glass modifier-trehalose mixture. Thus, thepresent invention encompasses the compositions and methods of making thecompositions comprised of trehalose and at least one modifier, whereinthe compositions have T_(g)s equal to or greater than the same compositeglasses of pure trehalose. Suitable active glass modifiers include, butare not limited to, proteins and other hydrated macromolecules. Suitableproteins include any physiologically acceptable protein and may be inertor a protein to be delivered therapeutically, i.e. a bioactive material.

It has also been found that bioactive materials soluble only in organicsolvents can be dried in trehalose from an organic/aqueous solvent togive a conformation that is now soluble in aqueous solvents. Methods ofmaking the dried material and compositions obtained thereby are providedby the invention. The bioactive material is dissolved in anorganic/aqueous solvent in combination with an effective amount oftrehalose and then dried. This gives a solid solution of the bioactivematerial in a trehalose glass which then readily dissolves in an aqueoussolution to give an aqueous suspension of the insoluble bioactivematerial. It has now been shown that the immunosuppressant cyclosporin A(which is insoluble in water and normally administered as an oilemulsion) in a solution of trehalose in a 1:1 ethanol:water mixture canbe dried to give a clear glass of trehalose containing cyclosporin A.This glass can be milled to give a free flowing powder which if added towater dissolves instantaneously to give a suspension of cyclosporin A inwater. If the solution dried contained a mixture of trehalose/trehaloseoctaacetate (insoluble in water), then the glass formed can be tailoredfor different dissolution rates by varying the ratio of the two.

Preferably, the compositions contain an amount of at least onephysiologically acceptable salt which effects a loss of water from thecomposition so that at ambient humidity the vapor pressure of water ofcrystallization is at least 14 mm Hg (2000 Pa) at 20° C. (molecularwater-pump buffer, hereinafter referred to as “MWPB”) and does notinterfere with glass formation of the stabilizing polyol. In the case ofpowders for pulmonary administration, addition of an effective amount ofMWPBs is particularly preferred as they have been found to preventwetting and clumping. An effective amount of an MWPB is one whichsubstantially prevents wetting and clumping. Suitable salts are thosedescribed in Spanish pat. no. 2009704. These may constitute a buffersystem or may replace a substantial amount of a component of the bufferin a conventional formulation. Suitable salts include, but are notlimited to, ammonium chloride, orthophosphate and sulfate; bariumchloride dihydrate; calcium lactate pentahydrate; copper sulfatepentahydrate; magnesium salicylate tetrahydrate, magnesium sulfateheptahydrate; potassium bisulfate, bromide, chromate and dihydrogenorthophosphate; sodium acetate trihydrate, bromoiridate dodecahydrate,carbonate decahydrate, fluoride, hydrogen orthophosphate dodecahydrate,metaperiodate trihydrate, metaphosphate trihydrate and hexahydrate,sulfite heptahydrate, sulfate heptahydrate and decahydrate andthiosulfate pentahydrate; and zinc sulfate heptahydrate and combinationsthereof.

Preferably, if the bioactive material and/or glass forming polyolcontain carboxyl and amino, imino or guanidino groups, the compositionsfurther contain at least one physiologically acceptable inhibitor of theMaillard reaction in an amount effective to substantially preventcondensation of amino groups and reactive carbonyl groups in thecomposition.

The inhibitor of the Maillard reaction can be any known in the art. Theinhibitor is present in an amount sufficient to prevent, orsubstantially prevent, condensation of amino groups and reactivecarbonyl groups. Typically, the amino groups are present on thebioactive material and the carbonyl groups are present on thecarbohydrate, or the converse. However, the amino and carbonyl groupsmay be intramolecular within either the biological substance or thecarbohydrate. Various classes of compounds are known to exhibit aninhibiting effect on the Maillard reaction and hence to be of use in thecompositions described herein. These compounds are generally eithercompetitive or noncompetitive inhibitors. Competitive inhibitorsinclude, but are not limited to, amino acid residues (both D and L),combinations of amino acid residues and peptides. Particularly preferredare lysine, arginine, histidine and tryptophan. Lysine and arginine arethe most effective. There are many known noncompetitive inhibitors.These include, but are not limited to, aminoguanidine and derivativesand amphotericin B.

EP-A-O 433 679 also describes suitable Maillard inhibitors which are4-hydroxy-5,8-dioxoquinoline derivatives.

As discussed below, the composition may further contain at least onephysiologically acceptable glass. Suitable glasses include, but are notlimited to, carboxylate, nitrate, sulfate, bisulfate, carbohydratederivatives and combinations thereof. Carboxylate and carbohydratederivatives are preferred where water soluble glasses are required asmany of these derivatives are slowly soluble in water. Suitable glassesinclude, but are not limited to, those described in PCT/GB 90/00497.

The composition may also be coated with one or more layers of aphosphate glass having a predetermined solution rate. The compositionmay further contain other water soluble and biodegradable glass formers.Suitable glass formers include, but are not limited to, lactide andlactide/glycolide copolymers, glucuronide polymers and other polyesters,polyorthoesters, and polyanhydrides.

In one embodiment, the delivery vehicle of the invention is sized andshaped for penetration of the epidermis and is suitable for ballisticdelivery. Suitable vehicle size is thus on the order of microns,preferably in the range of 1-5 microns in diameter and 5-150 microns inlength, which allows penetration and delivery through the epidermis tosubcutaneous and intradermal tissues. It will be appreciated that, atthis size, the delivery vehicle may macroscopically appear to be inpowder form, regardless of its configuration at the microscopic level.

Preferred configurations of the delivery vehicle of the invention aremicroneedles and microfibers of a stabilizing polyol glass. Themanufacture of microfibers is relatively simple and economical andresults in stable delivery vehicles comprised of the stabilizing polyoland the bioactive material. Additional stabilizers, buffers, glasses andpolymers may also be added as described herein. Many of the most labilebiomolecules can withstand high temperatures (e.g., 60-100° C.) whenstabilized by drying in trehalose, provided that the majority of theirsurface is in contact with the stabilizing polyol. Temperatures of 70°C. can be tolerated for over a month (Colaco et al. (1992)Bio/Technology 10:1007-1011) and higher temperatures for shorterperiods. The results presented herein show that the fluorescent proteinphycoerythrin dried in trehalose can be stored at 100° C. for at leastone month with no detectable loss of functional activity. Otherstabilizing polyols give protection at lower temperatures thantrehalose. The maximum temperature of protection must be determinedempirically and is within the skill of one in the art without undueexperimentation.

Providing the exposure time is limited, bioactive materials admixed indry stabilizing polyols can be heated to fluidize the glass which canthen be drawn or spun as a fiber without damage to the product. Fiberscan either be drawn from a billet and wound onto a drum or they can bespun through fine holes in a rapidly rotating cylinder that is heatedabove the melting point of the glass. Being inherently brittle, theseglass fibers can be readily crushed or chopped into short lengths toform long cylindrical rods or needles. By varying the diameter of thefibers produced, needles can be formed which vary from micro to macroneedles, i.e., from thicknesses of a few microns to fractions of amillimeter. It has been found that cotton candy machines are suitablefor use in preparing the microfibers. Although the optimal conditionsmust be determined empirically for each stabilizing polyol, suchdeterminations are well within the skill of one in the art.

The microfibers prepared in accord with the principles of the presentinvention, have a relatively high aspect ratio, i.e., length compared todiameter, preferably in the range of 1-5 microns in diameter and 5-150microns in length. This high aspect ratio provides for enhanced “end on”penetration upon ballistic delivery, by the tendency of the microfibersto lineup parallel to the barrel of the ballistic microinjector,described in more detail below. Longer macrofibers may be injected usingconventional impact ballistic devices or by trocar.

Alternative preferred embodiments of the delivery vehicle includeuniform microspheres, preferably with a narrow size distribution. Thisconfiguration is particularly useful when increased control of the depthof penetration of the delivery vehicle is desirable. Such control wouldbe useful, for example, for intradermal delivery of vaccines to thebasal layer of the epidermis, to bring antigen into proximity to theLangerhans cells of the skin to induce optimal immune responses.

To prepare microspheres of the present invention, several methods can beemployed depending upon the desired application of the deliveryvehicles. Suitable methods include, but are not limited to, spraydrying, freeze drying, air drying, vacuum drying, fluidized-bed drying,milling, co-precipitation and critical fluid extraction. In the case ofspray drying, freeze drying, air drying, vacuum drying, fluidized-beddrying and critical fluid extraction; the components (stabilizingpolyol, bioactive material, buffers etc.) are first dissolved orsuspended in aqueous conditions. In the case of milling, the componentsare mixed in the dried form and milled by any method known in the art.In the case of co-precipitation, the components are mixed in organicconditions and processed as described below. Spray drying can be used toload the stabilizing polyol with the bioactive material. The componentsare mixed under aqueous conditions and dried using precision nozzles toproduce extremely uniform droplets in a drying chamber. Suitable spraydrying machines include, but are not limited to, Buchi, NIRO, APV andLab-plant spray driers used according to the manufacturer'sinstructions. A number of carbohydrates are unsuitable for use in spraydrying as the melting points of the carbohydrates are too low causingthe dried materials to adhere to the sides of the drying chamber.Generally, carbohydrates with a melting point of less than the spraydrying chamber are unsuitable for use in spray drying. For example,palatinit and lactitol are not suitable for use in spray drying underconventional conditions. A determination of suitable carbohydrates canthus be made on known melting points or determined empirically. Suchdeterminations are within the skill of one in the art.

An alternative method for manufacturing microspheres as deliveryvehicles in accord with the present invention is to prepare a uniformaqueous/organic phase emulsion of the bioactive material in a solutionof the stabilizing polyol as the aqueous phase and the glass former inthe organic phase. This is followed by drying of the emulsion dropletsto form a solid solution of the bioactive material and stabilizingpolyol in an amorphous matrix of the glass former. In a modification ofthis method, the emulsion may be formed from the bioactive compound insolid solution in the stabilizing polyol and two different polymersdissolved together in one solvent, or dissolved into two separatesolvents. The solvent(s) are then removed by evaporation to yield doubleor multi-walled microspheres. Suitable methods for making multi-walledmicrospheres are described, for instance, in Pekarek et al. (1994)Nature 367:258-260; and U.S. Pat. No. 4,861,627.

The bioactive material can also be dried from an organic solution of thestabilizing polyol and the bioactive material to form a glass containinghomogeneously distributed bioactive material in solid solution in thepolyol glass. These glasses can then be milled and/or micronized to givemicroparticles of homogeneous defined sized.

The bioactive material and the stabilizing polyol can also beco-precipitated to give high quality powders. Co-precipitation isperformed by spraying, for instance with an air brush, the bioactivematerial and stabilizing polyol and/or glass former into a liquid inwhich neither dissolves, such as ice-cold acetone.

The invention also encompasses hollow fibers for delivery of bioactivematerials. By drawing down a heated hollow billet, fine hollow needlescan be formed. These can be made to contain a finely powdered stabilizedcompound by introduction of the fine powder during the melting anddrawing down process. The hollow fiber can also be made ofthermoplastic, organic polymer and/or carbohydrate and/or derivatizedcarbohydrate glass which may itself be water soluble or biodegradable.

An alternative embodiment of the delivery vehicle in the inventioncomprises a hollow vehicle comprised of water soluble glass or plasticwhich is filled and optionally coated with stabilizing polyol glass andthe bioactive material. Fine hollow fibers of water-soluble inorganic ororganic glasses can be drawn from a hollow billet and a finely powderedstabilizing polyol-bioactive material can be incorporated into the lumenof the billet, and therefore of the fiber, during the process.Alternatively, hollow needles of these glasses may be filled by allowingcapillarity to draw up suspensions of the finely powdered bioactivesubstance in a volatile organic solvent which is subsequently removed byevaporation leaving the needle filled with the bioactive substance. In amodification of this method, incorporation of a soluble glass former inthe organic solvent phase will result in the needle being filled withthe bioactive substance in solid solution in the glass former.

In another embodiment of the invention, coformulations of stabilizingpolyol glass and other water soluble materials are included. Forexample, coformulations of stabilizing polyol glass with water-solubleglasses such as phosphate glasses (Pilkington Glass Company) orbiodegradable plastics such as lactide or lactide/glycolide copolymerswill yield a more slowly eroding vehicle for delayed release of thebioactive material. A finely powdered stabilizing polyol glass/bioactivematerial can be intimately mixed with a finely powdered carboxylateglass and co-sintered. Alternatively, if a metal carboxylate glass has alower melting point than the stabilized bioactive polyol glass, thelatter can be homogeneously embedded as a solution in a carboxylateglass by cooling the melt obtained. This can be milled to give a finepowder with solubilities intermediate between the rapid solubility ofthe stabilizing polyol and the slow solubility of the carboxylate glass.

Alternate coformulations include the use of a homogeneous suspension ofthe finely powdered bioactive material/stabilizing polyol mixtureencapsulated in a carboxylate glass by drying from an organic solutionof the carboxylate to form the carboxylate glass. This can the ground togive a fine powder which would have the rapidly dissolving stabilizingpolyol glass containing the encapsulated bioactive material entrappedwithin a slow dissolving carboxylate glass (i.e., a conventionalslow-release system). Pulsatile release formats can be achieved eitherby repeated encapsulation cycles using glasses of different dissolutionrates, or by mixing powders of a number of coformulations with thedesired range of release characteristics. Note that this glass couldalso be drawn or spun to give microfibers or microneedles which would beslow-release implants. It will be appreciated that any stabilizingpolyol formulation should be such that it is capable of releasing thebioactive material upon administration, and should not unduly effect thestability of the material being administered.

As discussed above, glasses of derivatized carbohydrates are alsosuitable for use herein. Suitable derivatized carbohydrates include, butare not limited to, carbohydrate esters, ethers, imides and other poorlywater-soluble derivatives and polymers.

The delivery vehicle is loaded with the bioactive materials to bedelivered to the tissue by drying a solution of the bioactive materialcontaining a sufficient quantity of stabilizing polyol to form a glasson drying. This drying can be accomplished by any method known in theart, including, but not limited to, freeze drying, vacuum, spray, belt,air or fluidized-bed drying. The dried material can be milled to a finepowder before further processing the material with the polyol glass orcoformulation.

Different dosing schemes can also be achieved depending on the deliveryvehicle employed. A stabilizing polyol glass delivery vehicle of theinvention can provide for a quick release or flooding dose of thebioactive material after administration, upon the dissolving and releaseof the bioactive material from the stabilizing polyol glass.Coformulations of stabilizing polyol with water soluble glasses andplastics such as phosphate, nitrate or carboxylate glasses andlactide/glycolide, glucuronide or polyhydroxybutyrate plastics andpolyesters, can provide more slowly dissolving vehicles for a slowerrelease and prolonged dosing effect. A booster effect can also berealized by utilizing a hollow water soluble vehicle filled and coatedwith a stabilizing polyol glass loaded with the bioactive material. Thepolyol glass coating loaded with the material will dissolve rapidly togive an initial dosing effect. While the hollow outer portion of thevehicle dissolves, there will be no dosing action, followed by a boostereffect of the inner filling comprised of a stabilizing polyol and abioactive material when the hollow outer portion is breached bydissolution. Such pulsatile release format is particularly useful forvaccine delivery. Should multiple effect pulsatile delivery bedesirable, delivery vehicles with any combination of layers of watersoluble “non-loaded” materials and stabilizing polyol glass loaded withthe bioactive material can be constructed.

The delivery of more than one bioactive material can also be achievedusing a delivery vehicle comprised of multiple coatings or layers of thestabilizing polyol loaded with different materials or mixtures thereof.Administration of the solid dose delivery vehicle of the presentinvention can be used in conjunction with other conventional therapiesand coadministered with other therapeutic, prophylactic or diagnosticsubstances.

The invention further encompasses methods of delivery. Suitable deliverymethods include, but are not limited to, topical, transdermal,transmucosal, oral, gastrointestinal, subcutaneous, ocular, andby-inhalation (naso-pharyngeal and pulmonary, including transbronchialand transalveolar). Topical administration is, for instance, by adressing or bandage having dispersed therein the stabilizing polyolglass/bioactive material, or by direct administration into incisions oropen wounds. Creams or ointments having dispersed therein slow releasebeads of bioactive material/stabilizing polyol are suitable for use astopical ointments or wound filling agents.

Compositions for transdermal administration are preferably powders ofmicroneedles or microbeads. Larger, macroscopic needles and beads arealso provided for subdermal implantation and extended drug delivery. Theparticle sizes should be small enough so that they do not cause skindamage upon administration. Preferably, the powders are microneedles ofapproximately 10-1,000 microns in length and 1-150 microns in diameter.The powders may be prepackaged in single-dose, sealed, sterile formats.Suitable methods of transdermal administration include, but are notlimited to, ballistic, trocar and liquid jet delivery. Ballisticadministration is preferred as it is relatively painless. Generally thedelivery vehicle is accelerated in a shock wave of helium or another gasand fired into the epidermis. A suitable device for ballistic deliveryis described in PCT/GB 94/00753. A suitable device for liquid-jetdelivery is a Medi-ject device (Diabetes Care (1993) 16, 1479-1484).Such liquid-jet devices are particularly useful with the largermacroneedle delivery vehicles which may also be delivered by the use ofconventional impact ballistic devices or by trocar.

Upon transdermal administration, the degree of penetration of thedelivery vehicle can be controlled to a certain degree, not only by theballistic microinjector, described below, but also the shape and size ofthe powder particles. For example, when a relatively uniform and lesserdegree of penetration is desirable, microspheres may be more suitablefor the practice of the present invention. When a greater degree ofpenetration is desirable, a microfiber configuration may be preferred.Because the aspect ratio (i.e., length to diameter) of the microneedlesis high they have higher masses than spherical particles with a similardiameter. If they can be induced to impact with the skin “end-on,” theirhigher mass will give them a higher momentum for the same velocity andthey will thus penetrate deeper into the tissues. When randomlyorientated microneedles are put into a laminar flow of gas, they willalign themselves in the direction of the air flow and in thegas-propelled ballistic injector this will ensure that they impact theskin at the right angles and thus penetrate it.

The compositions suitable for transmucosal delivery include, but are notlimited to, lozenges for oral delivery, pessaries, and rings and otherdevices for vaginal or cervical delivery.

Compositions suitable for gastrointestinal administration include, butare not limited to, pharmaceutically acceptable powders and pills foringestion and suppositories for rectal administration.

Compositions suitable for subcutaneous administration include, but arenot limited to, various implants. Preferably the implants aremacroscopic spherical or cylindrical shapes for ease of insertion andmay be either fast or slow release. Since the entire implant isdissolved in the body fluids, removal of the implant is not necessary.Furthermore, the implants do not contain synthetic polymers and thus areless likely to initiate a separate immune response.

Compositions suitable for ocular administration include, but are notlimited to microsphere and macrosphere formulations, and saline drops.

Compositions suitable for by-inhalation administration include, but arenot limited to, powders of bioactive material/stabilizing polyol.Preferably the powders are of a particle size 0.1 to 10 microns. Morepreferably, the particle size is 0.5 to 5 microns. Most preferably,particle size is 1 to 4 microns. In particular for pulmonaryadministration, the preferred particle size is 2.5-3 microns. Preferablythe powders also contain an effective amount of a physiologicallyacceptable MWPB. An effective amount of an MWPB is one whichsufficiently reduces wetting to prevent substantial clumping, forinstance, a 50% molar ratio of potassium sulfate. Sodium sulfate andcalcium lactate are the preferred salts with potassium sulfate being themost preferred. Atomizers and vaporizers filled with the powders arealso encompassed by the invention.

There are a variety of devices suitable for use in by-inhalationdelivery of powders. See, e.g., Lindberg (1993) Summary of Lecture atManagement Forum, Dec. 6-7, 1993 “Creating the Future for PortableInhalers.” Additional devices suitable for use herein include, but arenot limited to, those described in WO9413271, WO9408552, WO9309832 andU.S. Pat. No. 5,239,993.

The following examples are provided to illustrate but not limit thepresent invention.

EXAMPLE 1 Methods of Making Solid Dose Delivery Vehicles

a) Carbohydrate Glass Microfiber Formation.

Glasses were formed by drying 20% solutions of either trehalose,lactitol, palatinit or GPS, containing MWPB and 1 mg/ml of thefluorescent algal protein phycoerythrin under vacuum (80 mtorr) for 16hrs. The glasses were ground in a domestic coffee mill to yield a coarsepowder which was used to fill the spinning head of a Kando K1 KandyFloss cotton candy machine (GB Patent No. 00103/76). The motor was thenswitched on and the powdered sugar glass heated at element settingsbetween 5 and 9. Residence time in the spinning head was 2-10 min and acontinuous process was maintained by constantly topping up the head.

The fibers produced were ground in a domestic coffee grinder and theresults obtained are presented in Table 1, which shows an average-of theneedles produced. These data indicate that, with all three sugarglasses, reduced element settings result in the production of finerdiameter microneedles. With trehalose, setting 6 gave microneedles witha mean diameter of 15 microns, and setting 9, microneedles with a meandiameter of 40 microns. With GPS, setting 9 gave microneedles with amean diameter of 15 microns. Microneedles formed from glasses containingbuffer salts remained dry at ambient temperatures and humidities.Microneedles containing phycoerythrin showed retention of biologicalactivity as assessed by fluorescence.

TABLE 1 Microneedle size analysis Length (μm) Width (μm) Mean 192.6043.35 Standard Error 12.53 2.33 Median 167.5 37.5 Mode 137.5 47.5Standard Deviation 123.44 22.91 Sample Variance 15237.75 524.72 Kurtosis16.17 2.55 Skewness 3.35 1.45 Range 862.5 115 Minimum 67.5 10 Maximum930 125 Sum 18682.5 4205 Count 97 97 Confidence Level (95.000%) 24.574.56

b) Binary Carbohydrate/Organic Mixture Glass Microfiber Formation.

Glasses were formed by drying a 5:1:1 mixture of trehalose, sodiumoctanoate and water under vacuum (80 mTorr) for 16 hrs. The glasses wereground in a domestic coffee mill to yield a coarse powder which was usedto fill the spinning head of a Kando K1 Kandy Floss machine. The motorwas then switched on and the powdered binary carbohydrate/organic glassheated at element settings between 5 and 9. As with pure trehaloseglasses, reduced element settings resulted in the production of finerdiameter microneedles. The binary mixture glasses can be tailored toyield glasses with significantly different tensile properties comparedto the corresponding pure trehalose glasses. Residence time in thespinning head was again 2-10 min and a continuous process was maintainedby constantly topping up the head. The results obtained indicate thatvariations of the melting points and dissolution times of the glassesand the resulting physical properties of the microfibers can be achievedby varying both the carbohydrate/organic molecules and ratios used.

EXAMPLE 2 Methods of Making Solid Dose Delivery Vehicles

a) Micronized Powder Preparation.

Glasses were formed by drying 20% solutions of either trehalose,lactitol, palatinit, GPM or GPS, containing an equimolar ratio of MWPBand protein, by freeze-drying under vacuum (80 mTorr) for 16 hrs. Theglasses were powdered using a Trost air-jet mill. Particle size in themicronized powders were measured using a Malvern Mastersizer laserparticle sizer. The results obtained with micronized powders obtainedfrom an original solution of 0.5 M trehalose and 0.5 M calcium lactateshowed a monodisperse particle distribution with mean particle diametersof 1.1 microns (FIG. 1). The powders containing MWPB remained afree-flowing powder and showed no change in particle size or clumpingand uptake of water on extended exposure to ambient temperatures andhumidities (FIGS. 2A and 2B).

b) Spray-Dried Powder Preparation.

20% solutions of trehalose containing MWPB salts and protein(phycoerythrin) were dried in a Buchi or Lab-Plant spray drier at a pumpspeed of 500-550 ml/hr and an inlet temperature of 180° C. Particle sizewas again measured using a SympaTec laser particle sizer. Thespray-dried powders showed a monodisperse particle distribution with asufficiently narrow peak size distribution for effective use asparticles in a powder ballistic device. In the results shown in FIG. 3,particle size analysis of a spray-dried powder produced by spray dryinga mixture of 0.5 M trehalose and 0.5 M calcium lactate on a Lab-Plantspray drier showed a mean particle diameter of 8.55 microns andillustrates the tight peak distribution obtained. Variation of the meanparticle size can be achieved by varying either the composition of themixture to be spray dried or the characteristics of the spray driernozzle assembly used. The results shown in FIG. 4 provide a comparisonof the particle-size analysis of the spray-dried powder as in FIG. 3with a spray-dried powder produced by drying the same mixture on theBuchi spray drier which uses a different nozzle assembly. The peakdistribution shown in FIG. 4 shows an equally narrow range but the meanparticle size is now 7.55 microns. These data show that the particlesobtained by different spray-drying processes are equally suitable toprovide compositions for ballistic delivery. Note that the ability tovary particle size results in compositions with different penetrativecharacteristics. This is particularly important for determiningintradermal or intramuscular delivery as the penetration is a functionof particle momentum and the distribution is a function of the scatterof particle size.

c) Drying from Organic Solvents

A 50 mg/ml solution of cyclosporin A in a 1.1 mixture of ethanol:water,containing 20% trehalose, was air-dried at ambient temperature to form aclear trehalose glass containing cyclosporin A in solid solution. Theglass was ground to give a powder, according to the method described inExample 1, and remained a free-flowing powder at ambient temperature andhumidities. Addition of the powder to water resulted in the dissolutionof the trehalose and the formation of a uniform aqueous suspension ofcyclosporin A.

d) Co-Precipitation Powder Preparation

20% solutions of trehalose, lactitol, palatinit, GPM or GPS, containingMWPB and protein (phycoerythrin) were dried by spraying into anacetone-solid carbon dioxide freezing bath. The precipitated powderswere separated by centrifugation or filtration and air dried to removeresidual solvent. The powders again showed a monodisperse particledistribution and those containing buffer formulation salts remained dryat ambient temperatures and humidities.

EXAMPLE 3 Variable Solubility of Glasses of Carbohydrate/CarbohydrateEster Coformulations

Various ratios of trehalose and trehalose octaacetate (TOAC) or twodifferent carbohydrate esters were dissolved in pyridine with sufficientwater added to give a clear solution. The solutions were dried rapidlyto give clear transparent monophasic glasses of the carbohydrate and/orcarbohydrate ester mixes. TOAC is almost insoluble in water andincreased amounts of the ester in the mixture resulted in the increaseddissolution times of the coformulated glass formed.

Coformulations of TOAC and raffinose undecaacetate containing 1-2%Mordant Blue (MB9) dye were prepared as described above. The releaserates of MB9 were measured by absorbance quantitatedspectrophoto-metrically and the results are depicted in FIG. 5. Theseresults indicate that glasses of two carbohydrate derivatives providedifferent release characteristics and that the use of two or morecarbohydrate derivatives results in glasses tailored to provide desiredrelease characteristics.

EXAMPLE 4 Protection of Proteins Against an Organic Solvent and ElevatedTemperatures Effected by Drying in Trehalose

a) Protection of Horseradish Peroxidase and Alkaline Phosphatase AgainstAcetone Effected by Drying in Trehalose

A 0.1 mg/ml horseradish peroxidase solution or a 1 mg/ml alkalinephosphatase/4 mg/ml bovine serum albumin Solution was dried in an FTSSystems freeze drier with or without 50% trehalose. The drier was usedas a vacuum drier and the mixtures dried without freezing. Four timesthe volume of solvent was added and the solution was allowed toevaporate to dryness. The contents were redissolved in 5 milliliters ofwater, and enzyme activity was assessed, in serial dilution, bycommercial ‘kit’ reagents. The alkaline phosphatase kit was obtainedfrom Sigma Chemical Co. and the horseradish peroxidase kit was obtainedfrom Kirkegaard & Perry Laboratories, Inc. As shown in FIGS. 6A and 6B,the enzymes dried with trehalose were more resistant to acetone than theenzymes dried without trehalose.

b) Protection of Phycoerythrin Against Organic Solvents Afforded byDrying In Trehalose

A 400 μg/ml phycoerythrin solution was freeze-dried in a Labconcofreeze-drier with or without 20% trehalose. The dried protein powder wasexposed to a number of organic solvents for 72 hrs. The phycoerythrinremained fluorescent in acetone, acetonitrile chloroform and methanol.In pyridine, the phycoerythrin remained fluorescent for 24-48 hr butbegan wetting and lost fluorescence by 72 hrs. In dimethylsulfoxide, thepowder solubilized but the phycoerythrin remained fluorescent.

c) Protection of Phycoerythrin Against 100° C. Afforded by Drying inTrehalose

A 400 μg/ml phycoerythrin solution was freeze-dried in the FTS drierwith or without 20% trehalose. The dried protein was stored at 100° C.for one month with no loss of functional activity.

d) Effect of Protein on T_(g) of Trehalose

The presence of protein in a powdered trehalose glass has now been foundto stabilize the glass against the plasticizing effected by water onpure trehalose glasses. This is illustrated by the results depicted inFIG. 7, which show the effect of water on the glass transitiontemperature of trehalose glasses with (solid line) or without (brokenline) bovine serum albumin at concentrations of from 0.002-50%. Thiseffect is not seen or is seen only partially with other carbohydrates,as illustrated by the results depicted in FIG. 8 utilizing maltose.

This elevation of T_(g) by protein is utilized to formulate trehalosestabilized protein in a pure trehalose glass. A powderedprotein-containing trehalose glass is prepared as described in Example1, added to the melt of a pure trehalose glass and the mixtureimmediately quenched to give the trehalose-stabilized protein powder ina solid solution in a pure trehalose glass. This glass can then befurther processed as described in Examples 1 and 2. A similar embeddedglass can be formed if an alternative stabilizing polyol with a T_(g)lower than that of trehalose is used to form the pure sugar glass, whichagain allows this glass to be melted and used below the melting point ofthe powdered, stabilized-protein glass to be embedded. For example,palatinit glasses melt at 60-70° C. at which temperature theprotein-stabilized powder is still a glass and the trehalose-stabilizedprotein glass can thus be encapsulated in the palatinit glass melt bysimply mixing and quenching.

EXAMPLE 5 Preparation of Bioactive Material/Stabilizing PolyolCompositions

a) Microparticles of trehalose containing MB9 were prepared by spraydrying as described in Example 2b. The solution dried contained 0.39 Mtrehalose and 0.14 M calcium lactate and 0.5% MB9. These particles werecoated by adding them to a saturated solution of zinc palmitate (ZnCl₁₆)in toluene and cooling from 60° C. to 30° C. This deposited a layer ofZnCl₁₆ on the particles which were then filtered under pressure toremove the excess ZnCl₁₆, washed with acetone and air-dried. Theresulting powder remained unwetted in water for at least three days (theparticles floated in the water without sinking or releasing MB9 andthereafter slowly released dye into the water). Thus, otherwise watersoluble powders may be made water impermeable by coating with metalcarboxylates such as ZnCl₁₆ to yield slow release formats. Note that thecoating material is most likely in crystalline form and not a glass;therefore, the solid phase in which the bioactive materials aresuspended need not be in the glass phase to be impermeable.

b) Coformulation of Carbohydrate and Organic Glasses by Evaporation

A powdered trehalose glass containing phycoerythrin was added to a 1:1mixture of sodium octanoate and zinc ethylhexanoate dissolved in anexcess of chloroform and evaporated under a stream of N₂ at roomtemperature to yield a carboxylate glass containing phycoerythrin powderin solid solution. The coformulated glass remained insoluble in waterfor at least 48 hrs. The phycoerythrin powder remained fluorescent bothin the initial organic solution and in the final glass.

c) Coformulation of Carbohydrate and Organic Glasses by Co-Melting

A preformed organic glass formed by quenching a melt of 1:1 mixture ofsodium octanoate and zinc ethylhexanoate was melted at 95° C. and apowdered trehalose glass containing phycoerythrin was added to the melt.The resultant mixture was immediately quenched on an aluminum blockprecooled to 15° C. A clear carboxylate glass formed containingencapsulated phycoerythrin powder which retained its biologicalfunctionality as assayed by its ability to fluoresce. Varying the natureand ratios of the carbohydrate and organic moieties in the coformulatedglasses results in glasses with a range of slow-release characteristicsas assessed from their variable dissolution times in water.

d) Coformulation of Carbohydrate Glasses and Plastics by Evaporation

A powdered trehalose glass containing phycoerythrin prepared accordingto Example 1 was added to a solution of perspex filings dissolved in anexcess of chloroform and evaporated under a stream of N₂ at roomtemperature to yield a solid perspex block containing the phycoerythrinpowder in solid solution. The phycoerythrin powder remained fluorescentboth in the initial organic solution and in the reformed solid perspexwhich was impermeable to water even after 4 weeks. Similar results wereobtained with polyester dissolved in dichloromethane and polyurethanedissolved in dimethylsulfoxide.

EXAMPLE 6 Preparation of Hollow Needles Filled with Bioactive Materials

The end of a billet of a trehalose glass tubes with a central cavityfilled with a powdered trehalose glass containing phycoerythrin preparedaccording to Example 1 was melted in a zone furnace and the fiber drawnby winding onto a metal drum rotated at constant speed. The hollowfibers formed contain the finely powdered trehalose-stabilized compoundand can be cut to any desired size. The hollow fiber can also be made ofthermoplastic, organic glass or carbohydrate which may itself be watersoluble, and by varying the diameter of the fibers produced, the filledneedles can be formed which vary from micro to macro needles, i.e. fromthicknesses of microns to fractions of a millimeter. The hollow needlesmay be filled with any solid dose vehicle described herein.

EXAMPLE 7 Ballistic Delivery of Solid Dosage Delivery Vehicle

Powdered glasses were injected into the skin by propulsion at hypersonicspeeds using a pressure shock wave created by the release of compressedgas. The powder was held in the chamber attached to the large end of afunnel-shaped cavity to the smaller end of which was attached acartridge of compressed gas sealed by a mylar film and the hypersonicshock wave was generated by rupture of the mylar membrane.Alternatively, a timer relay-driven solenoid can be used to control thehelium release which would allow functioning at lower helium pressures.This is the principle used in the particle inflow gun (PIG) developed byFiner for transforming plant tissues. Vain et al. (1993) Plant CellTissue and Organ Culture 33:237-246.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

1. A therapeutic composition in solid dose form comprising a bioactivematerial and a carbohydrate, wherein a) the bioactive material is aprotein, b) the carbohydrate stabilizes the bioactive material in thepresence of an organic solvent, c) the therapeutic composition comprisestherapeutic particles which contain said protein homogeneouslydistributed in solid solution in said carbohydrate in a glassy state, d)the therapeutic composition is a powder having a particle sizedistribution suitable for pulmonary administration, e) the therapeuticparticles containing said protein and said carbohydrate in the solidsolution remain in the glassy state when stored at 60° C. for one month,f) the carbohydrate is selected from the group consisting ofmonosaccharides, disaccharides and sugar alcohols, and g) thetherapeutic particles have a mean particle size of 0.5 to 5 μm.
 2. Thetherapeutic composition according to claim 1, said therapeutic particlesfurther comprising a physiologically acceptable inhibitor of theMaillard reaction.
 3. The therapeutic composition according to claim 1,said therapeutic particles additionally comprising a biodegradableglass.
 4. The therapeutic composition according to claim 1, wherein theparticles have a mean particle size of 1 to 4 μm.
 5. The therapeuticcomposition according to claim 1, wherein the carbohydrate is a sugaralcohol.
 6. The therapeutic composition according to claim 1, whereinthe carbohydrate is trehalose.
 7. The therapeutic composition accordingto claim 1, wherein the protein is insulin.
 8. The therapeuticcomposition according to claim 7, wherein the particles have a meanparticle size of 1 to 4 μm.
 9. The therapeutic composition according toclaim 1, wherein the protein is insulin and the carbohydrate istrehalose.
 10. The therapeutic composition according to claim 9, whereinthe particles have a mean particle size of 1 to 4 μm.
 11. Thetherapeutic composition according to claim 1, said therapeutic particlesfurther comprising an amino acid that is capable of inhibiting theMaillard reaction.
 12. The therapeutic composition according to claim 1,wherein the organic solvent is selected from the group consisting ofethanol, acetone, chloroform, acetonitrile, dichloromethane, andmethanol.
 13. The therapeutic composition according to claim 1, whereinthe therapeutic composition is suitable for delivering the bioactivematerial by transalveolar administration.
 14. A therapeutic compositionin solid dose form comprising a bioactive material and a carbohydrate,wherein a) the bioactive material is a protein, b) the bioactivematerial of the therapeutic composition is stabilized in the presence ofan organic solvent, c) the therapeutic composition comprises therapeuticparticles which contain said protein homogeneously distributed in solidsolution in said carbohydrate in a glassy state, d) the therapeuticcomposition is a powder having a particle size distribution suitable forpulmonary administration, e) the therapeutic composition furthercomprises a glass modifier, wherein the glass modifier is not thebioactive material, f) the therapeutic particles containing the proteinand the carbohydrate in the solid solution remain in the glassy statewhen stored at 60° C. for one month, g) the carbohydrate is selectedfrom the group consisting of monosaccharides, disaccharides and sugaralcohols, and h) the therapeutic particles have a mean particle size of0.5 to 5 μm.
 15. The therapeutic composition according to claim 14,wherein the glass modifier is a protein.
 16. The therapeutic compositionaccording to claim 14, said therapeutic particles further comprising aphysiologically acceptable inhibitor of the Maillard reaction.
 17. Thetherapeutic composition according to claim 14, said therapeuticparticles additionally comprising a biodegradable glass.
 18. Thetherapeutic composition according to claim 14, wherein the particleshave a mean particle size of 1 to 4 μm.
 19. The therapeutic compositionaccording to claim 14, wherein the carbohydrate is a sugar alcohol. 20.The therapeutic composition according to claim 14, wherein thecarbohydrate is trehalose.
 21. The therapeutic composition according toclaim 14, wherein the protein is insulin.
 22. The therapeuticcomposition according to claim 21, wherein the particles have a meanparticle size of 1 to 4 μm.
 23. The therapeutic composition according toclaim 14, wherein the protein is insulin and the carbohydrate istrehalose.
 24. The therapeutic composition according to claim 23,wherein the particles have a mean particle size of 1 to 4 μm.
 25. Thetherapeutic composition according to claim 14, said therapeuticparticles further comprising an amino acid that is capable of inhibitingthe Maillard reaction.
 26. The therapeutic composition according toclaim 14, wherein the organic solvent is selected from the groupconsisting of ethanol, acetone, chloroform, acetonitrile,dichloromethane, and methanol.
 27. The therapeutic composition accordingto claim 14, wherein the therapeutic composition is suitable fordelivering the bioactive material by transalveolar administration. 28.The therapeutic composition according to claim 1, said therapeuticparticles additionally comprising a water soluble glass.
 29. Thetherapeutic composition according to claim 28 wherein the organicsolvent is dichloromethane or chloroform.
 30. The therapeuticcomposition according to claim 7, said therapeutic particlesadditionally comprising a water soluble glass.
 31. The therapeuticcomposition according to claim 1, said therapeutic particlesadditionally comprising a water soluble glass.
 32. The therapeuticcomposition according to claim 31 wherein the organic solvent isdichloromethane or chloroform.
 33. The therapeutic composition accordingto claim 1, said therapeutic particles additionally comprising a watersoluble glass.
 34. The therapeutic composition according to claim 1,wherein the therapeutic composition is suitable for delivering thebioactive material by transalveolar administration.
 35. The therapeuticcomposition according to claim 34, wherein the carbohydrate is a sugaralcohol.
 36. The therapeutic composition according to claim 35, saidtherapeutic particles further comprising an amino acid that is capableof inhibiting the Maillard reaction
 37. The therapeutic compositionaccording to claim 14, wherein the therapeutic composition is suitablefor delivering the bioactive material by transalveolar administration.38. The therapeutic composition according to claim 37, wherein thecarbohydrate is a sugar alcohol.
 39. The therapeutic compositionaccording to claim 38, said therapeutic particles further comprising andamino acid that is capable of inhibiting the Maillard reaction.